Guys,
Here is a study that was done on peptides. They are fine at room temp for quite a while.
DETAILED DESCRIPTION OF THE INVENTION
European Patent EP0909177
The present invention is drawn to the unexpected discovery that dissolving high concentrations (i.e., at least 10%) of peptide compounds in water results in stable aqueous formulations. Previously known aqueous formulations of peptide compounds, which are dilute buffered aqueous solutions containing excipients such as EDTA or ascorbic acid which must be stored at low temperatures (4-25°C), form degradation products using degradation pathways such as acid/base catalyzed hydrolysis, deamidation, racemization and oxidation. In contrast, the presently claimed formulations stabilize peptide compounds at high concentrations at elevated temperatures (e.g., 37°C to 80°C), thus making possible the delivery of peptides in implantable delivery devices that would not otherwise be feasible.
Standard peptide and protein formulations consist of dilute aqueous solutions. Two critical aspects of peptide formulation include solubilization and stabilization of the drug molecule. Peptide stability is usually achieved by varying one or more of the following: pH, buffer type, ionic strength, excipients (EDTA, ascorbic acid, etc.). In contrast, in the present invention, highly concentrated peptides formulated in water provide stable solutions.
The invention consists of using high concentrations of peptide in aqueous solution to stabilize the peptide formulations against both chemical and physical degradation.
Preparation of Formulations
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...the concentration of peptide compound may range from at least 10% (w/w) to the maximum solubility of the compound. A preferred range is from 20 to 60% (w/w). The currently more preferred range is from 30 to 50% (w/w) and a most preferred range is 35 to : 45% (w/w).
Generally, the stable formulations of the present invention may be prepared by simply dissolving a therapeutically effective amount of the desired peptide compound in water, although pH adjustments may be made.
Methodology
We have found that stable aqueous formulations of peptide compounds may be prepared by dissolving a high concentration (at least about 10%) of the peptide compound to be formulated in water.
We have tested these peptide compound formulations, specifically formulations of the LHRH-related compound leuprolide, for stability by subjecting them to accelerated aging at elevated temperature and measuring the chemical and physical stability of the formulations. Results of these studies (shown, for example, in Table III and Figures 1, 2 and 6) demonstrate that these formulations were stable at conditions that approximate or exceed storage for one year at 37°C.
We have also tested peptide compound formulations prepared as described herein for stability after 2.5 megarad gamma irradiation. Results. shown in Table IV, show that these formulations remained chemically and physically stable after such irradiation. Formulations subjected to electron beam irradiation were also found to be stable.
As shown in Table I, we have tested a wide variety of peptide formulations, specifically leuprolide, goserelin, LHRH, angiotensin I, bradykinin, calcitonin, insulin, trypsinogen and vasopressin, for stability by dissolving (or attempting to dissolve) them in water, then subjecting them to accelerated aging at elevated temperatures. The stability of the formulations was measured. Results are presented in Table I as half-life at 37°C assuming an E a = 22.2 kcal/mole. A wide range of the peptides tested were soluble in water and remained stable under the test conditions. The solubility of a particular peptide in water and the stability of the resulting solution are easily determined using routine procedures known to those of ordinary skill in the art.
Formulations of 40% leuprolide in water stored for six months at 37°C showed linear degradation as measured by overall loss of peptide from the solution. Analysis of these data gave an activation energy (E a ) of 22.2 kcal/mole and a t 90 of 13.8 months, showing stability of these formulations at elevated temperatures.
We have also unexpectedly found that certain peptide formulations of the present invention are bacteriostatic (i.e., inhibit bacterial growth), bactericidal (i.e., cause the death of bacteria), and sporicidal (i.e., kill spores). In particular, leuprolide formulations of 50-400 mg/ml exhibited bacteriostatic, bactericidal and sporicidal activity. The stability of the samples was unaffected by spiking with bacteria, indicating that the enzymes released from the killed and lysed bacteria did not adversely affect the stability of the product. This demonstrates that these formulations were not conducive to enzymatic activity.
Some peptides, for example calcitonin and leuprolide, are known to be physically unstable, exhibiting aggregation, gelation and fibrillation when formulated in aqueous solution. For example, leuprolide can be induced to gel by increasing peptide concentration, introduction of salts or gentle agitation. Improving physical stability can allow for easier parenteral administration, including administration using implantable drug delivery systems.
It has unexpectedly been found that adding polar aprotic solvents such as DMSO to aqueous formulations of certain peptides, such as leuprolide, goserelin and calcitonin, prevents gelation of the formulation. This is apparently because non-aqueous polar aprotic solvents cause peptides to form a random coil/alpha helix conformation that does not refold into a beta sheet structure and, therefore, does not gel. Thus, these solvents have an anti-gellant effect.
Additionally, studies of gelled and non-gelled aqueous formulations of leuprolide (370 mg/ml) stored at 37°C for 6 weeks showed a similar chemical stability profile as assayed by RP-HPLC. Results are shown in Figure 8. Similarly, the stability of liquid and gelled (by agitation) aqueous leuprolide formulations (370 mg/ml) was studied in vitro at 37°C and in vivo in rats, respectively. Results are presented in Table II, and show that the both gelled and liquid formulations remained stable over a period of 18 weeks.
A major aspect of the invention is that aqueous solutions containing high concentrations of peptide compounds are stable at high temperatures for long periods of time. Thus, these formulations are advantageous in that they may be shipped and/or stored for long periods of time at or above room temperature. They are also suitable for use in implantable delivery devices.
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Examples of peptides and peptide compounds which may be formulated using the present invention include those peptides which have biological activity or which may be used to treat a disease or other pathological condition. They include, but are not limited to adrenocorticotropic hormone, angiotensin I and II, atrial natriuretic peptide, bombesin, bradykinin, calcitonin, cerebellin, dynorphin A, alpha and beta endorphin, endothelin, enkephalin, epidermal growth factor, fertirelin, follicular gonadotropin releasing peptide, galanin, glucagon, gonadorelin, gonadotropin, goserelin, growth hormone releasing peptide, histrelin, insulin, leuprolide, LHRH, motilin, nafarelin, neurotensin, oxytocin, somatostatin, substance P, tumor necrosis factor, triptorelin, and vasopressin. Analogs, derivatives, antagonists, agonists and pharmaceutically acceptable salts of the above may also be used.
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